J Biosci (2019) 44:38 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-019-9856-8 (0123456789().,-volV)

(0123456789().,-volV)

Review

Restriction enzymes and their use in molecular biology:

An overview

FRANCESCA DI FELICE1, GIOACCHINO MICHELI2 and GIORGIO CAMILLONI1,2*

1
Dipartimento di Biologia e Biotecnologie, Sapienza, Università di Roma, Piazzale A. Moro 5, 00185 Roma,
Italy
2
Istituto di Biologia e Patologia Molecolari, CNR, Roma, Piazzale A. Moro 5, 00185 Roma, Italy

*Corresponding author (Email, giorgio.camilloni@uniroma1.it)

MS received 24 October 2018; accepted 3 January 2019; published online 8 April 2019

Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the
molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence
specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization,
mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in
the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing
active witnesses of major steps in the field.

Keywords. Cloning; DNA manipulation; history; restriction enzymes

1. Introduction
Historia magistra vitae: Cicero’s expression, drawn from his
almost 2000-year-old De Oratore, brilliantly synthesizes how
the understanding of present reality must value past experience.
This requirement is highly relevant to scientific studies
and is nicely exemplified by the references every scientific
article relies on: new knowledge banks on previous acqui-
sitions and acknowledges them. In this light, we present an
overview on the experimental significance of a specific class
of proteins, type II restriction enzymes. It has been more
than half a century since the first studies on these molecules.
Their fundamental role as tools for the characterization and
manipulation of DNA quickly made them very well known
within the scientific community. Highlighting their relevance
to DNA studies goes in parallel with the history of the
double helix and may appeal also to those who, while not
working on DNA, are nonetheless interested in the pro-
gresses made by investigations in this area.
2. Restriction and modification
Major observations in microbial genetics revealing the
capability of bacteria to resist bacteriophage infections were
published in the early 1950s (Luria and Human 1952;
Bertani and Weigle 1953). These works describe the changes
the infectious capacity of the phage undergoes during the
growth cycle, showing that these alterations are not due to de
novo mutations or selective processes. Subsequently, it was
ascertained that the underlying defense system is based on
DNA sequence-specific endonucleases, which restrict viral
action (hence the term restriction enzymes) by digesting
phage DNA, coupled to a corresponding DNA methyl-
transferase activity, which safeguards the integrity of host
DNA by modifying potential cutting sites through the addi-
tion of methyl groups (reviewed in Wilson and Murray
1991).
For their seminal contribution to the discovery and use of
restriction enzymes, Werner Arber, Dan Nathans and
Hamilton Smith were awarded the 1978 Nobel Prize for
Physiology and Medicine. Arber had hypothesized that these
enzymes were able to bind DNA at sites represented by
specific DNA sequences (Arber 1965). Smith had verified
Arber’s hypothesis by using purified enzymes and showing
that they were able to cut symmetric and specific nucleotide
sequences (Kelly and Smith 1970; Smith and Wilcox 1970).
Nathans had realized the possible exploitation of these
enzymes, producing the first restriction cutting site maps of
specific DNA fragments (Danna and Nathans 1971). Since
Arber’s, Smith’s and Nathans’ studies, the characterization
of restriction enzymes has developed very rapidly and four

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38 Page 2 of 8 F Di Felice, G Micheli and G Camilloni
different enzyme classes have been identified. The enzymes
belonging to class II are commonly exploited in manipu-
lating and characterizing DNA. They only require Mg2?,
and no ATP, to recognize their target DNA sequence and
cleave it. Many restriction enzymes, originating from a wide
range of bacterial species, belong to this class and hundreds
of recognition/cutting sequences have been identified. Using
different combinations of restriction enzymes, many differ-
ent ways of characterizing and manipulating DNA have
become possible (Williams 2003). Comparative analyses
have shown that type II restriction enzymes are members of
a large superfamily of proteins, called PD-(D/E)XK nucle-
ases, characterized by a conserved core fold (a common a/b
motif) but lacking significant sequence similarity except for
critical residues of their active site (Knizewski et al. 2007).
This poses interesting and as-yet open questions about the
evolutionary pathways of the many members of this family
(Gupta et al. 2012).
Class I, III and IV restriction enzymes are much less
frequently employed in DNA studies because of their lower
DNA-cutting specificity. Rather than coinciding with their
recognition sequences, their cutting sites often localize far
away and/or are not sequence specific. In addition, their
restriction activity is not always separated from the methy-
lation activity and their number is not as abundant as that of
class II restriction endonucleases. Finally, the activity of
class I, III and IV restriction enzymes is often ATP-depen-
dent (reviewed in Roberts et al. 2003).
3. Basic use of restriction enzymes: physical DNA
mapping
Since the first experiments by Danna and Nathans (1971),
the major use of restriction enzymes was aimed at locating
their cutting sites on selected DNA molecules. In an era
where effective nucleotide sequence determination methods
were yet under development, the specificity of the restriction
cleavage profiles of different DNA regions allowed to easily
compare them, resulting in an unprecedented flow of highly
interesting structural, functional and evolutionary studies on
many gene systems (Holsinger and Jansen 1993). Compar-
ing DNAs from organisms belonging to different, even
closely related taxa became as simple as comparing their
restriction profiles. Indeed, a single nucleotide variation
occurring at a given restriction cutting site is sufficient to
affect cleavage and result in a different digestion pattern. A
breakthrough in physical mapping of DNA by restriction
enzyme digestion came about in 1976 with the introduction
of the Southern blot approach. In essence, the experimental
scenario was standardized by Edwin Southern’s method to
analyze specific DNA restriction fragments after sorting
them electrophoretically by size through an agarose gel slab
(Southern 1975). The possibility to analyze only a subset of
fragments was of particular interest: the DNA was denatured
within the gel at the end of the electrophoretic separation and
the entire gel content was then transferred (blotted) onto a
flexible membrane (usually a nitrocellulose filter) so as to
make the DNA fragments bind to this support without
altering the relative positions they had reached in the gel.
The filter was then subjected to molecular hybridization with
specific, radioactively labeled DNA probes. Thus, only the
fragment(s) corresponding to the probe were evidenced.
A particularly interesting application of restriction map-
ping is based on the analysis of restriction fragment-length
polymorphisms (RFLP). This technique allows to compare
DNA samples drawn from different sources (individuals) by
evaluating the size variability of specific restriction frag-
ments. Fragments from a given DNA region of a single
individual are compared with a reference sample or scored
for an individual signature. RFLP analysis has gained wide
acceptance as a highly accurate tool in prenatal diagnostics.
The use of specific probes against single copy sequences,
capable to distinguish sequence polymorphisms when
hybridized to restriction endonuclease digested DNAs, has
been proposed for the first time during the 1980s (Botstein
et al. 1980; Weatherall et al. 1985). Later, this approach was
also widely adopted by forensic molecular genetics (Sajan-
tila and Budowle 1991; Balazs 1992). Nowadays conven-
tional forensic serology has been almost completely replaced
by DNA-based assays.
4. Basic use of restriction enzymes: DNA manipulation
The many heuristic and applicative approaches employing
restriction enzymes have proved fundamental for physical
DNA mapping. Similarly, recombinant DNA technology,
which has equally strong ties with these extraordinary
molecular tools, had a revolutionary impact on molecular
biology as well as on biomedicine and biotechnology.
Shortly before the identification of the first restriction
enzymes, Lederberg (1952) proposed to use the term ‘plas-
mid’ for any extrachromosomal element determining
heredity or sex. A few years later (Hickson et al. 1967) the
physical and chemical properties of plasmid DNA and its
circular nature were extensively characterized and plasmids
were also visualized by electron microscopy. In 1972 Cohen
and coworkers inserted an exogenous closed-circular DNA
harboring sequences encoding the resistance against a given
antibiotic into a bacterial strain. They selected the plasmid-
containing population by screening for the ability to grow in
the presence of the same antibiotic (Cohen et al. 1972).
At that time, the capacity of DNA ligase to join two
adjacent nucleotides aligned on a complementary template
by creating a new phosphodiester bond had been demon-
strated following experiments with E. coli extracts capable to
join polydeoxynucleotide chains and convert hydrogen
bonded circles from k-phage into a covalently closed-cir-
cular form (Cozzarelli et al. 1967; Gefter et al. 1967; Gellert
1967; Olivera and Lehman 1967; Weiss and Richardson
1967). However, at the beginning of the 1970s, a tool for the
 Restriction enzymes
specific fragmentation of DNA was still missing. Stanley
Cohen, one of the major personalities in the field, had been
experimenting with mechanical DNA fragmentation (Cohen
et al. 1967) but the right kind of highly specific ‘molecular
scissors’ became available only through the studies of Arber,
Smith and Nathans (Arber 1965; Smith and Wilcox 1970;
Danna and Nathans 1971). Work from Herbert Boyer’s lab
represented a landmark by providing an historical restriction
enzyme, EcoRI (Yoshimori et al. 1972). It also became clear
that some class II activities produced a staggered cut in the
double helix (figure 1), leaving complementary (the so-
called sticky or cohesive) ends (Hershey et al. 1963; Gellert
1967), which could be bridged, regardless of the origin of
the DNA, with DNA ligase (Jensen et al. 1971; Jackson
et al. 1972; Lobban and Kaiser 1973). Suddenly, the
recombinant DNA playground was ready. An excellent
description about the origins and the development of
molecular cloning, including an account of the famous
meeting at Waikiki beach (Hawaii), has been published in
recent years by Stanley Cohen (Cohen 2013).
By joining DNA fragments from different organisms, the
generation of the so-called chimeric DNAs became possible.
The insertion of some X. laevis rDNA fragments into the
pSC101 plasmid was one of the first examples (Morrow
et al. 1974). These experiments proved that it was possible
to use bacterial plasmids to clone DNA from various sour-
ces; that the junction of DNAs from different organisms
could take place after cutting them with restriction enzymes
generating the same type of ends; and, last but not least, that
this procedure did not affect the functionality of the plasmid
itself which continued replicating and transcribing the har-
bored genes.
Then, another important step followed: the creation of
gene libraries (reviewed in Durmaz et al. 2015). These
libraries involved genomic DNA fragmentation by digestion
with restriction enzymes cutting with high frequency, i.e.
having short recognition sequences (4–6 bases). The frag-
ments obtained were joined in vitro to a number of plasmid
molecules ensuring statistically sufficient coverage of the
whole genome. Thus, restriction fragments obtained from a
given genome were distributed on different plasmids which
collectively represented the whole genome of the organism.
These libraries have allowed a systematic study of entire
genomes, even those with a remarkably large size (e.g. the
human one). Overall, the use of restriction enzymes to split
large DNA chunks into fragments of defined size and with
specific ends has paved the way not only to recombinant
DNA technology but also and to the first DNA sequencing
efforts (reviewed in Heather and Chain 2016).
Restriction enzyme-mediated manipulation of DNA has
opened the possibility to introduce targeted deletions of gene
or promoter sub-regions, in order to compare the behavior of
deleted templates with wild-type copies in terms of sub-
strates for RNA transcription/processing and translation. The
ability to cut and join gene pieces almost at will has provided
tremendous momentum to basic knowledge on the nature,
Page 3 of 8 38
function and regulation of genes, and has led to remarkable
biotechnological achievements. It became possible to deeply
engineer genes in vitro, even human ones, transcribe them
and give rise, by subsequent translation, to proteins of
medical interest such as globins or insulin. The latter was
produced for the first time in 1979 (Goeddel et al. 1979).
Since then, the production of complex, biologically active
molecules by means of recombinant DNA technologies has
become common practice (Khan et al. 2016).
These findings have greatly stimulated the research on
site-specific manipulation of the genome, with emphasis on
the development of endonuclease-based tools able to target
and cleave virtually any sequence. This has led to two
powerful systems: ZFN (zinc-finger nuclease) (Liu et al.
1997) and TALEN (transcription activator-like effector
nuclease) (Christian et al. 2010; Miller et al. 2011). ZFN
relies on artificial nucleases where the DNA-cleavage
domain of Fok I, a type II restriction enzyme, is fused to the
C-terminal of a zinc-finger DNA binding domain: in the
fusion protein the Fok I nuclease domain is responsible for
the cutting activity, whereas the zinc-finger domain recog-
nizes and binds the target sequence. The TALEN system also
exploits a fusion protein, constituted by the Fok I cleavage
domain and a transcription activator-like effector DNA-
binding domain (Gaj et al. 2013). Until 2011 ZFN and
TALEN were the most promising systems endowed with
high targeting/cutting specificity. However, in terms of time
and economical requirements both systems have proven
rather demanding and a substantial progress occurred with
the introduction of programmable RNA-mediated target-
ing/cutting systems (Kim and Kim 2014) as RGEN (RNA-
guided engineered nucleases), which has been rapidly
replaced by its more effective, and now widely adopted
successor, CRISPR-Cas9 (see Section 6).
5. DNA accessibility studies: chromatin, DNA
methylation
The study of chromatin structure has been largely based on
nuclease resistance analyses (Hewish and Burgoyne 1973;
Rill and Van Holde 1973; Noll 1974) that led to the basic
nucleosome model proposed by R. Kornberg (Kornberg
1974). The nuclease approach was followed by another
methodology (REAA, restriction enzyme accessibility assay)
based on the cutting specificity of restriction enzymes to test
the in vivo accessibility of specific DNA regions (Pfeiffer
et al. 1975; Hörz et al. 1976; Lipchitz and Axel 1976).
Successive reports showed that transcribed genes were more
sensitive to restriction enzymes than non-transcribed ones
(Grummt and Gross 1980). Cutting was hindered when the
restriction site mapped within a DNA region associated with
histones to form a nucleosome. Conversely, if the location of
the nucleosome did not encompass the restriction site, cut-
ting could occur. Thus, it became possible to observe
nucleosome positioning or chromatin remodeling events
38 Page 4 of 8 F Di Felice, G Micheli and G Camilloni

Figure 1. Diagram showing the general mechanism of action of type II restriction endonucleases and the end products of the reaction. In
the presence of Mg2? ions a type II enzyme catalyzes the cleavage of phosphodiester bonds (solid arrows pointing at the bond between 30 O
and P) at specific locations along the DNA, generating fragments with 50 -phosphoryl/30 -hydroxyl ends. The reaction occurs by nucleophilic
attack at the phosphorus atom, but it is as yet not fully established whether it proceeds by direct hydrolysis or through the formation of a
covalent reaction intermediate (Pingoud et al. 2005). While individual chemical steps of the actual mechanism may exhibit specific
variations for each restriction enzyme, the general scheme reported here is well conserved. The nucleotide sequence shown (GAATTC) is
the recognition/cleavage site for EcoRI, a restriction enzyme which operates a staggered cut leaving 50 -protruding, cohesive ends.

mediated, for instance, by environmental changes. REAA
has been used for a long time to evaluate changes in chro-
matin structure.
In 1978 Edwin Southern and Adrian Bird developed a
mapping strategy involving the DNA methylation status
(Bird and Southern 1978). In Xenopus laevis, ribosomal
DNA from erythrocytes (somatic rDNA) showed higher
resistance to the action of specific restriction enzymes as
compared to the same substrate abundantly present in
oocytes as amplified rDNA. The difference turned out to
depend on the differential methylation status of the rDNA in
the two tissues rather than on different nucleotide sequences.
This approach gave rise to a number of comparisons
exploiting differential methylation sensitivity to restriction
enzymes and represents the origin of molecular epigenetics,
which subsequently gained enormous impulse with the
advent of high yield DNA sequencing (Lin 2018).
6. CRISPR-Cas9: new frontiers in DNA editing
The ability of restriction endonucleases to distinguish
between methylated and non-methylated DNA substrates, is
essential in safeguarding the bacterial genome against bac-
teriophage invasion. Restriction enzymes are regarded as a
pivotal component of an innate defense system developed by
 Restriction enzymes
Figure 2. Simplified diagram of CRISPR–Cas9 as an RNA-
driven DNA targeting/cleavage system. A synthetic small, single-
guide RNA (sgRNA) carries a short sequence (usually 20
nucleotides; gray dotted line) able to match with a target DNA
site (black dotted line). Target recognition is facilitated by the
presence of a short sequence (50 -NGG), the protospacer-adjacent
motif (PAM). Upon complementary base paring and R-loop
formation the Cas9 endonuclease is activated and scission (arrows)
of both DNA strands occurs (see Jiang and Doudna 2017 for a
recent in-depth review).
Figure 3. Restriction enzymes: active witnesses of DNA history.
Page 5 of 8 38
bacteria during evolution. An additional defense mechanism
exploited by bacteria has recently stirred great interest. It is
based on CRISPR elements (clustered regularly interspected
short palindromic repeats) and on their associated proteins,
Cas. At each phage infection, fragments from the phage
genome are integrated into the bacterial genome at CRISPR
loci (Barrangou et al. 2007). These loci are then transcribed
and small RNAs originating from the integrated phage DNA
fragments recognize the exogenous sequences. These RNAs
are used as a guide by a specific Cas protein, Cas9 (CRISPR
associated protein 9), an endonuclease encoded by a gene
located near the CRISPR loci. When the RNA-guide pairs
with the phage DNA sequence, Cas9 is activated and
degrades phage DNA.
Both defense mechanisms, restriction/modification and
CRISPR-Cas9, exploit endonucleolytic activities. The
effectiveness of the first mechanism relies on the ability of
the restriction endonuclease to distinguish and selectively
inactivate phage DNA, while preserving host DNA, on the
basis of the methylation status. The second mechanism
(figure 2) takes advantage of the RNA guide forming a
RNA/DNA duplex with phage DNA, and thus inducing the
Cas9-mediated cleavage of the targeted sequence (this can
occur even later, during a subsequent infection by the same
phage).
Similarly to restriction enzymes, albeit at a much quicker
pace, also the CRISPR-Cas9 system has undergone exten-
sive studies to clarify the underlying molecular mechanisms
and appropriately engineer them to manipulate DNA
38 Page 6 of 8 F Di Felice, G Micheli and G Camilloni
Figure 4. Timeline showing major milestones in the history of restriction enzymes.
templates. In essence, the CRISPR-Cas9 system has quickly
turned into a novel and very powerful biotechnological tool,
making genome editing virtually possible in any cell. This is
exemplified by the possibility to introduce genetic mutations
at specific locations by simply identifying an appropriate/
unique target DNA sequence and adopting the correspond-
ing RNA guide to bring the Cas9 endonuclease in position
(Jinek et al. 2012). Such a high specificity would not be
achievable with restriction endonucleases as their recogni-
tion/cutting sites are dispersed all over the genome. Cutting
sites of type II restriction enzymes are ‘hard wired’ into the
structure of the protein, i.e. changing the recognition/cutting
specificity requires a new protein. Conversely, the Cas9
endonuclease relies on a small guide RNA to pair with its
complementary DNA target and a new guide RNA is suffi-
cient to change the cutting specificity, without the need to
modify the protein.
7. Concluding remarks
Since their discovery restriction enzymes have been widely
used in fundamental DNA technology approaches like DNA
cloning, mapping and manipulation. The availability of a
molecular tool able to generate specific DNA fragments has
been a turning point in the history of DNA. The involvement
of restriction enzymes in a wide variety of sophisticated
procedures makes these proteins active ‘molecular wit-
nesses’ of the progresses in the analysis, manipulation and
exploitation of the double helix (figure 3). What was initially
a discovery driven by the interests and principles of basic
research has rapidly become a powerful tool for applied
science and translational approaches (figure 4). Interestingly,
the CRISP/Cas system is currently following a closely
similar path. Overall, these observations constitute an
excellent exemplification of how basic, non-oriented
research represents a very valuable reservoir of new dis-
coveries with a strong applicative potential.
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